Bioprinting and regeneration of auricular cartilage using a bioactive bioink based on microporous photocrosslinkable acellular cartilage matrix

Tissue engineering provides a promising strategy for auricular reconstruction.Although the first international clinical breakthrough of tissue-engineered auricular reconstruction has been realized based on polymer scaffolds,this approach has not been recognized as a clinically available treatment because of its unsatisfactory clinical efficacy.This is mainly since reconstruction constructs easily cause inflammation and deformation.In this study,we present a novel strategy for the development of biological auricle equivalents with precise shapes,low immunogenicity,and excellent mechanics using auricular chondrocytes and a bioactive bioink based on biomimetic microporous methacrylate-modified acellular cartilage matrix(ACMMA)with the assistance of gelatin methacrylate(GelMA),poly(ethylene oxide)(PEO),and polycaprolactone(PCL)by integrating multi-nozzle bioprinting technology.Photocrosslinkable ACMMA is used to emulate the intricacy of the cartilage-specific microenvironment for active cellular behavior,while GelMA,PEO,and PCL are used to balance printability and physical properties for precise structural stability,form the microporous structure for unhindered nutrient exchange,and provide mechanical support for higher shape fidelity,respectively.Finally,mature auricular cartilage-like tissues with high morphological fidelity,excellent elasticity,abundant cartilage lacunae,and cartilage-specific ECM deposition are successfully regenerated in vivo,which provides new opportunities and novel strategies for the fabrication and regeneration of patient-specific auricular cartilage.

3D porous acellular cartilage matrix scaffold with surface mediated sustainable release of TGF-β3 for cartilage engineering

Acellular tissue matrix scaffolds are much closer to tissue’s complex natural structure and biological characteristics,thus assess great advantages in cartilage engineering.We used rabbit costal cartilage to prepare acellular microfilaments and further 3D porous acellular cartilage scaffold via crosslinking.Poly(_L-lysine)/hyaluronic acid(PLL/HA)multilayer film was then built up onto the surface of the resulting porous scaffold.Furthermore,TGF-β3 was loaded into the PLL/HA multilayer film coated scaffold to obtain a 3D porous acellular cartilage scaffold with sustained releasing of TGF-β3 up to 60 days.The success of this project will provide a new way for the treatment of articular cartilage defects.Meanwhile,the anchoring and on-site sustained releasing of growth factors mediated by polyelectrolyte multilayered film can also provide a new method for improving the biocompatibility and the biofunctionality for other implanted biomaterials.

Altered serum level of cartilage oligomeric matrix protein and its association with coronary calcification in patients with coronary heart disease

Background Cartilage oligomeric matrix protein （COMP） is mainly found in the skeletal system and vascular smooth muscle cells. Recent researches showed that it had a protective function on blood vessels and could also inhibit vascular calcification. We investigated the serum COMPs in coronary heart disease （CHD） patients, and the relationship between serum COMP and the calcification of coronary artery. Methods A total of 233 consecutive chest pain patients who first underwent coronary angiography followed by multi-slice computed to- mography （MSCT） within six months were recruited and divided into two groups according to the coronary angiography luminal diameter narrowing percentages： CHD group （diameter narrowing 〉 50%, n = 194） and control group （diameter narrowing 〈 50%, n = 39）. The Gen- sini score, Syntax score and coronary artery calcium score （CACs） were calculated. The serum COMP level was determined using ELISA. Results The levels of COMP were significantly higher in the CHD group than in the control group 155.7 （124.5-194.5） ng/mL vs. 128.4 （113.0-159.9） ng/mL, P = 0.019. There were no correlation between COMP, Gensini score, Syntax score, severity of coronary stenosis and the number of coronary artery with stenosis 〉 50%. The serum COMP was correlated with age （r = 0.294, P 〈 0.001）, fasting glucose （r = 0.163, P = 0.015）, HbAlc （r = 0.194, P = 0.015） and CACs （r = 0.137, P = 0.037）. Stepwise linear regression analysis showed that COMP level and age were independent predictors of CACs in the CHD patients （fl = 0.402, t = 2.612, P = 0.015; fl = 0.472, t = 3.077, P = 0.005）. Performance of COMP for predicting CHD was shown as area under curve （AUC）： 0.632, 95% CI： 0.549-0.715 and upper tertile CACs was AUC： 0.602, 95% CI： 0.5264）.678 in receiver operating characteristic （ROC） curve analysis. Conclusion Calcification of coronary artery was an independent predictor of serum COMPs.

Association between serum cartilage oligomeric matrix protein and coronary artery calcification in maintenance hemodialysis patients

Background Coronary artery calcification(CAC)is common in end-stage renal disease(ESRD)patients,and the extent of CAC is closely related to cardiovascular outcomes in ESRD patients.Cartilage oligomeric matrix protein(COMP),as a component of the vascular matrix,has been found to be an inhibitor of arterial calcification in basic studies.However,there is no clinical research on the correlation between COMP and CAC in maintenance hemodialysis(MHD)patients.The aim of this study was to explore the relationship between serum COMP levels and CAC and cardiovascular events in MHD patients.Methods Serum COMP levels were compared between 54 MHD patients and 66 healthy people.MHD patients were then divided into three groups according to the tertiles of the concentration of COMP level and were followed up for major adverse cardiac events(MACEs),which were defined as a combined end point of new onset angina pectoris,nonfatal myocardial infarction,heart failure,coronary artery revascularization,hospitalization due to angina pectoris and all-cause deaths.The CAC score was calculated based on computed tomography scans.Results The serum COMP level in MHD patients was significantly higher than that in the general population[984.23(248.43-1902.61)ng/mL vs.219.01(97.26-821.92)ng/mL,P<0.01].Serum COMP levels were positively correlated with CAC(r=0.313,P=0.021)and serum parathyroid hormone in MHD patients(r=0.359,P<0.01).Linear regression suggested that after adjusting for age,fasting blood glucose(Glu)and glycosylated hemoglobin(HbAlc),CAC score was an independent predictor in the final model for COMP level(β=0.424,t=3.130,P<0.01).The receiver operating characteristic(ROC)curve showed that COMP≥994 mg/mL had 68.0%sensitivity and 72.4%specificity for the prediction of severe CAC[area under the curve(AUC):0.674,P=0.030,95%CI:0.526-0.882].After a median follow-up of 16 months(8-24 months),there was no difference in the incidence rate of MACEs between the upper,middle and lower serum COMP groups.Conclusions Our study found that MHD patients have higher levels of circulating COMP than controls.The serum COMP level is positively correlated with CAC score and could be used as a biomarker of severe CAC in MHD patients.However,there is no obvious correlation between serum COMP levels and the incidence of cardiovascular events.

Injectable cartilage matrix hydrogel loaded with cartilage endplate stem cells engineered to release exosomes for non-invasive treatment of intervertebral disc degeneration

Low back pain,mainly caused by intervertebral disc degeneration(IVDD),is a common health problem;however,current surgical treatments are less than satisfactory.Thus,it is essential to develop novel non-invasive surgical methods for IVDD treatment.Here,we describe a therapeutic strategy to inhibit IVDD by injecting hydrogels modified with the extracellular matrix of costal cartilage(ECM-Gels)that are loaded with cartilage endplate stem cells(CESCs).After loaded with CESCs overexpressing Sphk2(Lenti-Sphk2-CESCs)and injected near the cartilage endplate(CEP)of rats in vivo,ECM-Gels produced Sphk2-engineered exosomes(Lenti-Sphk2-Exos).These exosomes penetrated the annulus fibrosus(AF)and transported Sphk2 into the nucleus pulposus cells(NPCs).Sphk2 activated the phosphatidylinositol 3-kinase(PI3K)/p-AKT pathway as well as the intracellular autophagy of NPCs,ultimately ameliorating IVDD.This study provides a novel and efficient non-invasive combinational strategy for IVDD treatment using injectable ECM-Gels loaded with CESCs that express Sphk2 with sustained release of functional exosomes.

Genetic analysis and serum level of cartilage oligomeric matrix protein in patients with pseudoachondroplasia

Background Pseudoachondroplasia （PSACH） is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein （COMP）.Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation.There is evidence that decreased serum COMP concentration may serve as a diagnostic marker in PSACH.Here, we investigated the role of this gene and the serum COMP concentration in Chinese patients with PSACH.Methods A family with three patients and a sporadic case were recruited.Genomic and phenotypic data were recorded.The diagnosis of PSACH was made on the base of clinical evaluation.The genomic DNA was extracted from peripheral blood leukocytes.The 8-19 exons and flanking intron-exon boundary sequences of COMP were amplified by polymerase chain reaction （PCR） and screened for mutation by direct DNA sequencing.Serum COMP concentrations of 4 patients and age-compatible control group of 20 unrelated healthy subjects were analyzed on the basis of an ELISA Kit for human cartilage oligomeric matrix protein.Results A deletion （c.1447-1455del） was identified in exon 13 in the sporadic case.The mean serum COMP concentrations of four patients （3.12±2.28） were significantly lower than those of control group （10.86±2.21, P 〈0.05）.There was no overlap in the distribution of serum COMP concentration between PSACH patients and controls.Conclusions Mutations in COMP gene are responsible for the PSACH.Serum COMP concentration may be suggested as an additional diagnostic marker to aid clinical findings in suspected cases of PSACH.

EDM1： a novel point mutation in cartilage oligomeric matrix protein gene in a Chinese family with multiple epiphyseal dysplasia

Background Multiple epiphysis dysplasia （MED） is a common skeletal dysplasia with a significant locus heterogeneity. In the majority of clinically defined.cases, mutations have been identified in the gene encoding cartilage algometric matrix protein （ COMP）. Methods Five patients were included in the study. Linkage analysis and mutation analysis of the COMP gene were conducted in the patients and their family members. Results We have identified a novel mutation in axon 14 of COMP gene in the family. Conclusions This mutation produced a severe MED phenotype with marked short stature, early onset osteoarthritis, and remarkable radiographic changes. Our results extended the range of disease-causing mutations in COMP gene and contributed more information about relationship between mutations and phenotype.

Upregulation of Cartilage Oligomeric Matrix Protein and Bone Morphogenetic Protein-2 May Associate with Calcific Aortic Valve Disease

Objective:Calcific aortic valve disease(CAVD)affects millions of elderly people,and there is currently no effective way to stop or slow down its progression.Therefore,exploring the pathogenesis of CAVD is very important for prevention and treatment.Cartilage oligomeric matrix protein(COMP)have important role in cell phenotype change.This study is aimed to confirm whether COMP participate in CAVD and try to find the possible mechanisms.Methods:Human aortic valve tissues from Nanjing First Hospital(CAVD group,n=20;control group,n=11)were harvested.The expression level of COMP was tested by western blot and immunohistochemistry.Dual immunofluorescence staining was used for locating COMP.Bone morphogenetic protein-2(BMP2)signalling were tested by western blot.The animal model was also used to detect COMP level by immunohistochemistry.Results:The results showed that the expression level of COMP was significantly increased in the calcific valve samples when compared with that of the control valve(P<0.05);COMP was expressed near the calcific nodules and co-localized with a-smooth muscle actin(a-SMA).The protein levels of BMP2 and p-Smads 1/5/9 were markedly more highly expressed in the CAVD group than the control group(P<0.05).Furthermore,immunofluorescence detection showed that COMP and BMP2 were co-located in calcific valves.Conclusions:The above results suggested that upregulation of COMP and BMP2 may be associated with aortic valve calcification and that COMP may become a potential therapeutic target in human CAVD.

Osteopontin inhibits osteoarthritis progression via the OPN/CD44/PI3K signal axis

Chondrocyte degeneration and extracellular matrix component loss are the primary causes of osteoarthritis(OA).OA can be treated by inhibiting chondrocyte degeneration and increasing extracellular matrix component secretion.Osteopontin(OPN),a multifunctional protein,has gained immense attention with regard to its involvement in OA.This study aimed to explore the therapeutic value and mechanism of action of OPN in OA treatment.Results of the histomorphological analysis revealed a worn-off OA cartilage tissue surface,cartilage matrix layer deterioration,and calcium salt deposition.Compared to that in normal chondrocytes,in OA chondrocytes,the OPN,CD44,and PI3K protein and mRNA expression was upregulated.Further,siOPN,rhOPN,and rhOPN plus LS-C179404 interfered with OA chondrocytes.As verified in mice,OPN directly inhibited the expression level of PI3K in OA chondrocytes by binding with CD44.Morphological analysis of the knee joints demonstrated that OPN effectively inhibited OA progression via the OPN/CD44/PI3K signal axis.In conclusion,OPN activates intracellular PI3K signaling molecules by binding to CD44 on the cell surface to cause downstream cascading effects,thereby delaying chondrocyte degeneration and reducing cartilage matrix component loss;therefore,OPN is a potential therapeutic agent for OA.

Diagnosis with Multiple Epiphyseal Dysplasia Using Whole-exome Sequencing in a Chinese Family

Multiple epiphyseal dysplasia （MED; EDMI, OMIM 132400; EDM2, OMIM 600204; EDM3, OMIM 600969; EDM4, OMIM 226900; EDM5~ OMIM 607078; EDM6, OMIM 614135） is an autosomal dominant inherited disease of the skeletal system, characterized by mild short stature and early-onset degenerative joint disease, caused by heterogeneous genotypes involving more than six genes （COMP, COL9A 1, COL9A2, COL9A3, MATN3, DTDST）.However, in approximately 10-20% of all samples analyzed, a mutation cannot be identified in any of the six genes mentioned above, suggesting that the presence of other unidentified causative genes is also involved in the pathogenesis of MED.

The role of hyaluronic acid in biomineralization

Hyaluronic acid has been extensively investigated due to intrinsic properties of natural origin and strong ability to bind ions in water. Hyaluronic acid is an excellent crystal modifier because its abundant negatively charged carboxyl groups can bind the cations protruding from the crystal lattice. In this review, we mainly present the latest work focus on the role of hyaluronic acid in controlling the crystallization, breaking the symmetry of crystal, and the surface funtionalization of nanocrystals.